Support of the Infectivity of Hepatitis Delta Virus ... - Journal of Virology

Support of the Infectivity of Hepatitis Delta Virus Particles by the Envelope Proteins of Different Genotypes of Hepatitis B Virus Natalia Freitas,a Kenji Abe,b Celso Cunha,c Stephan Menne,d Severin O. Gudimaa Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USAa; Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japanb; Medical Microbiology Unit, Center for Malaria and Tropical Diseases, Institute of Hygiene and Tropical Medicine, New University of Lisbon, Lisbon, Portugalc; Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC, USAd

ABSTRACT

This study examined how the envelope proteins of 25 variants of hepatitis B virus (HBV) genotypes A to I support hepatitis delta virus (HDV) infectivity. The assembled virions bore the same HDV ribonucleoprotein and differed only by the HBV variantspecific envelope proteins coating the particles. The total HDV yields varied within a 122-fold range. A residue Y (position 374) in the HDV binding site was identified as critical for HDV assembly. Virions that bound antibodies, which recognize the region that includes the HBV matrix domain and predominantly but not exclusively immunoprecipitate the PreS1-containing virions, were termed PreS1*-HDVs. Using in vitro infection of primary human hepatocytes (PHH), we measured the specific infectivity (SI), which is the number of HDV genomes/cell produced by infection and normalized by the PreS1*-MOI, which is the multiplicity of infection that reflects the number of PreS1*-HDVs per cell in the inoculum used. The SI values varied within a 160-fold range and indicated a probable HBV genotype-specific trend of D > B > E > A in supporting HDV infectivity. Three variants, of genotypes B, C, and D, supported the highest SI values. We also determined the normalized index (NI) of infected PHH, which is the percentage of HDV-infected hepatocytes normalized by the PreS1*-MOI. Comparison of the SI and NI values revealed that, while a particular HBV variant may facilitate the infection of a relatively significant fraction of PHH, it may not always result in a considerable number of genomes that initiated replication after entry. The potential implications of these findings are discussed in the context of the mechanism of attachment/entry of HBV and HDV. IMPORTANCE

The study advances the understanding of the mechanisms of (i) attachment and entry of HDV and HBV and (ii) transmission of HDV infection/disease.

H

uman hepatitis B virus (HBV) remains a significant pathogen, with approximately 400 million chronic carriers around the world. Chronic HBV infection is a main risk factor for developing hepatocellular carcinoma (HCC) (1, 2). A recent study demonstrated that the number of HBV virions in an inoculum per se determined the kinetics of HBV spread through the liver and the timing and magnitude of the immune response, as well as the outcome of infection, i.e., whether the infection became transient or chronic (3). These results indicated that the infectivity of the virions and the rate of virus spread throughout the liver may play a decisive role for the clinical outcome of an infection. A number of previous studies suggested that HBV-associated liver pathogenesis is genotype specific and reported differences between HBV genotypes in terms of the severity of induced liver disease, HCC incidence, responsiveness to antiviral therapy, and infectivity of HBV virions. Some of those reports appeared to be controversial (4–15). The current study examined in more mechanistic detail how the envelope proteins of HBV contribute to the infectivity of the virions. As the experimental model, we used virions of human hepatitis delta virus (HDV). HDV is a subviral agent of HBV that, in nature, coexists with its helper virus in infected livers and uses HBV envelope proteins to form HDV virions and enter hepatocytes through the HBV-specific receptor that interacts with specific sequences in the PreS1 domain of the L (large) envelope protein (16). We assembled in cell culture 25 different types of HDV virions, all of which bore the same HDV ribonucleoprotein (RNP) inside and differed only by the envelope proteins coating the viri-

June 2014 Volume 88 Number 11

ons. The envelope proteins analyzed in this study represent 25 different natural variants of HBV that belong to nine HBV genotypes, A to I (14, 17). It was found that HBV envelope proteins apparently were able alone to determine the number of infected hepatocytes and the number of genomes that successfully initiated HDV replication after entering a susceptible cell. It became apparent that HBV envelope proteins are not only involved in attachment and entry but also likely facilitate at least one of the immediate postentry steps. These novel findings advance the understanding of the mechanism of virus entry and, possibly, trafficking and suggest an additional virus life cycle step(s) that is likely regulated by HBV envelope proteins. MATERIALS AND METHODS LMS vectors. Using the sequences of the HBV variants listed in Table 1, 24 corresponding vectors were constructed to express the large (L), middle (M), and small (S) envelope proteins (LMS vectors). Each construct bears a fragment of the genome of a particular HBV variant inserted into XhoI/ XbaI sites of the pSVL vector (Pharmacia). The inserted fragment begins at the start codon of the L, includes the entire L open reading frame,

Received 4 February 2014 Accepted 14 March 2014 Published ahead of print 19 March 2014 Editor: G. McFadden Address correspondence to Severin O. Gudima, [email protected]. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.00346-14

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TABLE 1 HBV variants used in the study Name of varianta

Accession no.

Stage of HBV infection (reference)

A

A0 A1 A2 A3 A4

X02763 AB126580 AB205118 X51970 M57663

Unknown Acutec Chronicc Unknown Unknown

B

B1 B2 B3 B4

AB205121 AB205119 AB205122 D00330

Chronicc Chronicc Acutec Chronic (28)

C

C1 C2 C3 C4 C5

AB205125 AB205124 AB033550 NAb AF411411

Acutec Chronicc Unknown (28) Unknown Chronic (29)

D

D1 D2 D3 D5

AB205127 AB205126 V01460 NA

Chronicc (30) Chronicc Unknown Unknown

E

E1 E2 E3

AB205192 AY739675 AB032431

Chronicc (31) Unknown (32) Chronic (33)

F G H I

F1 G1 H1 I1

X69798 AF405706 AB205010 AB231908

Unknown (34) Chronic (35) Chronicc (36) Acutec (17)

HBV genotype

a The name of each variant reflects the HBV genotype and the number of a particular variant among the other variants of the same genotype in the collection of HBV sequences. b NA, accession number is not available, since the sequence of the indicated HBV variant was not previously deposited into NCBI database. Therefore, the complete amino acid sequences of the L envelope proteins of variants C4 and D5 are presented in Fig. 1. c K. Abe, personal communication.

and ends about 100 nucleotides downstream from the posttranscriptional regulatory element (PRE) (PRE spans positions 1151 to 1684 of HBV genotype A) (18–20). In the resulting LMS vectors, the mRNA for the L envelope protein is transcribed from the simian virus 40 (SV40) late promoter and the transcription of the M/S mRNA is driven by an authentic HBV promoter. Variants A1, B3, C1, and C5 are natural M⫺ mutants that do not express the M envelope protein. To express L, M, and S of variant A0, the plasmid pSVB45H (21) was used. The mutations in the LMS-C4 and LMS-B4 vectors (LMS vectors encoding the envelope proteins of variants C4 and B4, respectively) were introduced using the QuikChange II site-directed mutagenesis kit (Agilent Technologies) according to the instructions of the manufacturer. To introduce the mutation F(374)Y, which would change the sequence in the HDV binding site (HDV-BS) (22) of variant B4 from 368-VIWMIWF W-375 to 368-VIWMIWYW-375 (the numbering is according to HBV genotype A [20] and counts the residue at the very N terminus of the PreS1 domain as number 1), the following two oligonucleotides were used: 734-GTTATATGGATGATATGGTATTGGGGGCCAAGTCTG TACA-773 and 773-TGTACAGACTTGGCCCCCAATACCATATCA TCCATATAAC-734 (the changed coding triplet is underlined). To create the mutants C4m1 and C4m2, the LMS vector encoding the envelope proteins of variant C4 was used and either a single mutation,

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V(12)M (for C4m1), or a combination of the mutations N(7)K plus V(12)M (for C4m2) was introduced into the N terminus of PreS1. To make the V(12)M change, the following two oligonucleotides were utilized: 2865-CAATCCTCGACAAGGCATGGGGACGAATCTTTC2897 and 2897-GAAAGATTCGTCCCCATGCCTTGTCGAGGATTG2865. The primers 2848-ATGGGAGGTTGGTCTTCCAAGCCTCGA CAAGG-2869 and 2869-CCTTGTCGAGGCTTGGAAGACCAACCT CCCAT-2848 were used to introduce the mutation N(7)K. To make the combination of the two mutations N(7)K plus V(12)M, the V(12)M change was first introduced into the LMS-C4 vector, and the resulting construct was used then in order to insert the additional N(7)K change. To create the L⫺ (that does not express the L envelope protein) version of the LMS-D1 vector, the above-mentioned QuikChange II site-directed mutagenesis kit was used as well. The mutation was introduced using the following two oligonucleotides: ggatcgatccctcgac-2848-ACGGGGCAGAATCTTTCC-2865 and 2865GGAAAGATTCTGCCCCGT-2848-gtcgagggatcgatcc. The sequences of the vector are shown by lowercase letters. The changed position (2849 in the HBV sequence) is underlined. The initiation codon for the L protein was changed from AUG to ACG (ACG codes for Thr), which resulted in the amino acid substitution M(1)T at the very N terminus of the PreS1 domain. The resulting construct therefore is the L⫺M⫹S⫹ vector, expressing only the M and S envelope proteins (but not the L) of variant D1. Transfection. To assemble HDV virions coated with the envelope proteins of a particular HBV variant, Huh7 cells (a gift from Camille Sureau) were cotransfected with a 1:1 mixture of the pSVLD3 plasmid (to initiate HDV replication) and the corresponding LMS vector, using Fugene HD reagent (Roche) (21, 23). In vitro infection of PHH. Prior to infection, HDV virions were concentrated about 100-fold using polyethylene glycol (PEG) (Sigma). Infection of plated primary human hepatocytes (PHH) in 48-well plates (purchased from Life Technologies) was conducted in the presence of 5% PEG 8000 as previously described (21). Briefly, the PHH were maintained in serum-free fully defined Hepato-STIM culture medium that was supplemented with epidermal growth factor (EGF) (BD Biosciences). The infection medium, containing HDV and 5% PEG, was incubated with PHH for 8 h. After the incubation with PHH, the infection medium was replaced with Hepato-STIM–EGF medium that contained neither virus nor PEG. For the measurements of specific infectivity (SI), multiplicities of infection (MOI) of 10 to 20 total HDV genome equivalents (GE)/per average hepatocyte were employed. For the analysis of infected cells using the immunofluorescence procedure, MOIs of 30 to 100 HDV GE/cell were used. Infected hepatocytes were analyzed at day 9 postinfection. IP. Immunoprecipitations (IP) of HDV virions were performed using a previously described protocol (23). Rabbit polyclonal antimatrix antibodies (GenScript) were raised against the peptide spanning positions 91-IPPPASTNRQSGRQPTPISPPLRDSHPQAMQWNSTAF H-128 of the PreS region (HBV serotype adw2, genotype A), which includes the conserved matrix domain (underlined) (23, 24). Using specific software (mobyle.pasteur.fr/cgi-bin/portal.py?#forms::antigenic), it was predicted that the above-described peptide bears a single antigenic site (TPISPPLRDS), located in the PreS1 domain. Therefore, IP with the antimatrix antibodies was expected to exclusively target PreS1-HDVs (i.e., HDV virions that bear the PreS1 domain on their outer surfaces). Briefly, aliquots of the prepared HDV virus stocks were incubated first with antimatrix antibodies at 4°C overnight and then with 100 ␮l of Pansorbin suspension in phosphate-buffered saline (PBS) (Calbiochem) for the next 2 h on ice. Next, Pansorbin was sedimented by centrifugation at 13,000 rpm for 1 min. The resulting pellet was washed four times with ice-cold PBS containing 0.5% Nonidet P-40 (Fisher) and then used for RNA isolation. HDV-specific qPCR. Total RNA from Huh7 cells, Pansorbin-bound particles, PHH, or virions was isolated with TRI reagent (Fisher) and treated with DNase (Life Technologies) prior to quantitative real-time PCR (qPCR). The qPCR was conducted using the 7500 real-time PCR instrument (Applied Biosystems) as described previously (21, 25). The forward primer was 312-G

Journal of Virology

Infectivity of Hepatitis Delta Virus Particles

FIG 1 Complete amino acid sequences of the large (L) envelope proteins of HBV variants C4 and D5. The sequences of HBV variants C4 and D5 (see Table 1) used in the current study were not previously deposited in the NCBI database and therefore do not have accession numbers. The complete amino acid sequences of the corresponding large (L) envelope proteins were deduced from the nucleotide sequences and are presented in single-letter code. The number of amino acids in the L protein of each variant is shown. Compared to that of C4, the L of D5 does not contain 11 N-terminal amino acids of the PreS1 domain.

GACCCCTTCAGCGAACA-329, the reverse primer was 393-CCTAGCATC TCCTCCTATCGCTAT-360, and the TaqMan probe was 332-AGGCGCTT CGAGCGGTAGGAGTAAGA-357 (the numbering is according to Kuo et al. [26]). The reverse primer was also used for reverse transcription. The copy numbers were measured using a 10-fold dilution series of in vitro-transcribed and gel-purified genomic (G) unit-length HDV RNA standard (range, 20 to 200,000 genome equivalents [GE] of HDV) and considering 1 million HDV RNA molecules equal to 1 picogram of G RNA standard (21, 25). IF. The details of the immunofluorescence (IF) procedure are described elsewhere (21, 27). Briefly, PHH were fixed with 4% paraformaldehyde, washed twice with PBS, and permeabilized using 0.2% Triton X-100. Anti-delta antigen rabbit polyclonal and mouse monoclonal antibodies against human alpha-tubulin (Sigma) were used to stain PHH. Nuclear DNA was stained using DAPI (4=,6=-diamidino-2-phenylindole). Stained PHH were analyzed using an inverted Nikon TE2000-U microscope with a 20⫻ objective. For each HDV type, approximately 400,000 hepatocytes were assayed per infection. About 10 different fields of stained cells were photographed. Using staining for alpha-tubulin and for nuclear DNA, the total number of cells was quantified. Using staining for delta antigens, the number of HDV-infected hepatocytes was counted. The percentage of HDV-infected cells was calculated by dividing the number of delta antigen-positive cells by the total number of hepatocytes.

RESULTS

Sequences of HBV variants used to express the envelope proteins. Table 1 summarizes the 25 HBV variants used in this study, which belong to nine HBV genotypes (A to I) and were acquired at different stages of HBV infection (14, 17, 28–36). The sequences of variants C4 and D5 (Table 1) are absent from the NCBI database. Therefore, the entire amino acid sequences of the L proteins of these variants are presented in Fig. 1. None of the sequences tested in this study contained easily identifiable alterations, such as deletions, insertions, or rearrangements. Variants A1, B3, C1, and C5 are natural M⫺ mutants and, thus, can facilitate only the expression of the L and S proteins. Twenty-five constructs that express the envelope proteins of different HBV variants (LMS vectors) were used for the assembly of 25 different types of HDV virions. Each HDV type was assembled by cotransfection of Huh7 cells with the plasmid pSVLD3, which initiates HDV genome replication, and a particular LMS vector that expressed the envelope proteins of a single HBV variant. The HDV types were designated HDV-A1, HDV-B2, HDV-C3, etc., according to the HBV variant used (Table 2). Therefore, (i) each HDV type contained within the virion the same HDV RNP (the HDV RNA genome and approximately 200 copies of the delta antigen [␦Ag] [37]) and (ii) the


only difference between the assembled types of HDV was a unique envelope that contained the envelope proteins of a particular HBV variant. Furthermore, the differences that were anticipated to be observed in the assembly and/or infectivity of the above-described HDV types would reflect the differences in the functioning of the envelope proteins that belong to different HBV variants. Characterization of the concentrated HDV stocks. The results of quantitative analysis of the different concentrated HDV virus stocks are summarized in Table 2. The total yield of assembled HDVs refers to the sum of all categories of HDV virions regardless of the combination of envelope proteins in the virion. All secreted virions contained the S envelope protein, while only a fraction of virions bore the L envelope protein on their outer surface (Table 2) (16). The values of the total HDV yields varied within a 122-fold range. The highest yield was observed for variant A0, and the lowest for B4. Figure 2 represents the alignment of the HDV binding site (HDV-BS) sequences for all HBV variants examined. The HDV-BS is a small, 8-amino-acid-long cytosolic loop that is located between the third and fourth transmembrane domains and is therefore present in the L, M, and S proteins (16, 22). In the HDV-BS, each variant has unchanged all three tryptophans that were shown to be important for HDV assembly (22). Clearly, either of the highly conserved versions of the HDV-BS, AIWMMWYW and VIWM(M/I)WYW, may facilitate a considerable yield of HDV. Only B4 has a unique change, Y(374)F, resulting in VIWMIWFW, which correlates with the lowest efficiency of HDV assembly (Table 2). As expected, the created mutant B4m1, bearing the reverse change F(374)Y, supported an approximately 88-fold increase of total HDV yield, which is (i) 72.0% of the yield of HDV-A0 and (ii) the highest value among the variants of genotype B studied (Table 2). The significance of the Y(374) residue per se for HDV assembly was not recognized before (22, 38). The previously produced mutant, in which five amino acids that included the sequence 373-WYW-375 of the HDV-BS were replaced by the Lys-Leu sequence, displayed levels of synthesis and egress of the S protein comparable to that of the wild type and supported about a 10-fold-reduced level of HDV assembly (38). Based on that and taking into consideration that all three tryptophans important for HDV assembly were intact in the HDV-BS of B4, our interpretation was that Y(374) is involved in the regulation of HDV RNP binding to HDV-BS, and the presence of the hydroxyl group on Y (in contrast to F) appears to be important for HDV

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TABLE 2 Characterization of the concentrated virus stocks of different HDV typesa Total yield of HDV virionsc

Yield of PreS1*-HDVsd

HBV genotype

Name of HDV typeb

GE/␮l

% Yield

GE/␮l

% Yield

A

HDV-A0 HDV-A1 HDV-A2 HDV-A3 HDV-A4

1.13 ⫻ 107 5.27 ⫻ 106 5.27 ⫻ 105 6.23 ⫻ 105 1.21 ⫻ 106

100.0 46.5 4.7 5.5 10.7

3.18 ⫻ 106 1.38 ⫻ 106 1.03 ⫻ 105 1.36 ⫻ 105 3.21 ⫻ 105

28.0 26.3 19.5 21.8 26.5

B

HDV-B1 HDV-B2 HDV-B3 HDV-B4 HDV-B4m1e

2.64 ⫻ 106 1.67 ⫻ 106 1.38 ⫻ 106 9.27 ⫻ 104 8.16 ⫻ 106

23.3 14.7 12.2 0.8 72.0

5.97 ⫻ 105 5.32 ⫻ 105 1.52 ⫻ 105 1.16 ⫻ 104 2.01 ⫻ 106

22.7 31.9 11.0 12.6 24.6

C

HDV-C1 HDV-C2 HDV-C3 HDV-C4 HDV-C5 HDV-C4m1f HDV-C4m2g

3.19 ⫻ 106 6.83 ⫻ 106 7.85 ⫻ 105 5.27 ⫻ 106 4.98 ⫻ 105 1.04 ⫻ 107 3.46 ⫻ 106

28.2 60.3 6.9 46.5 4.4 91.9 30.5

8.69 ⫻ 105 1.62 ⫻ 106 1.06 ⫻ 105 5.97 ⫻ 105 5.86 ⫻ 104 1.44 ⫻ 106 4.72 ⫻ 105

27.2 23.6 13.4 11.3 11.8 13.8 13.7

D

HDV-D1 HDV-D2 HDV-D3 HDV-D5

1.59 ⫻ 106 8.83 ⫻ 105 1.51 ⫻ 106 2.53 ⫻ 106

14.1 7.8 13.3 22.3

5.07 ⫻ 105 1.96 ⫻ 105 4.00 ⫻ 105 7.43 ⫻ 105

31.8 22.1 26.5 29.4

E

HDV-E1 HDV-E2 HDV-E3

2.49 ⫻ 106 5.30 ⫻ 105 2.84 ⫻ 106

22.0 4.7 25.1

6.65 ⫻ 105 1.64 ⫻ 105 8.45 ⫻ 105

26.7 31.0 29.8

F G H I

HDV-F1 HDV-G1 HDV-H1 HDV-I1

8.71 ⫻ 106 7.53 ⫻ 105 7.58 ⫻ 106 1.16 ⫻ 106

76.9 6.7 67.0 10.3

2.38 ⫻ 106 1.71 ⫻ 105 1.89 ⫻ 106 3.61 ⫻ 105

27.3 22.7 24.9 31.0

a

A robust procedure for HDV assembly was developed to (i) ensure that technical factors (such as variations between transfections, etc.) would have no significant contribution to the HDV yields measured and (ii) allow us to achieve reproducible results when independent assemblies were performed. b The name of each type of assembled HDV indicates the particular HBV variant and the envelope proteins used for the assembly of this HDV type, as described in the text. c The total yield of HDV virions reflects the total number of HDV genome-containing virus particles and refers to the sum of all categories of HDV virions regardless of the combination of the envelope proteins in the virion. All secreted virions contained the S envelope protein, while only a fraction of virions bore the L envelope protein on their outer surface (16). Total yield was quantified using qPCR (21) and shows the number of HDV genome equivalents (GE) per 1 ␮l of 100-fold-concentrated HDV virus stock. %, calculations of the percentages of the total yields of different HDV types were done relatively to the total yield of HDV-A0 virions, which was the highest and therefore was used as 100%. d The yield of PreS1*-HDVs represents the number of HDV virions that were immunoprecipitated using the antimatrix antibodies (which predominantly but not exclusively precipitate the PreS1-containing HDVs [see the text]) per 1 ␮l of 100-foldconcentrated HDV stock. As described in Materials and Methods, after immunoprecipitation with the antimatrix antibodies, the total RNA was then extracted from precipitated viral particles with TRI reagent and HDV genomes were quantified using qPCR (21). %, the portion of the PreS1*-HDVs in the total population of the assembled and released HDV particles. The immunoprecipitation procedure was optimized in order to achieve its maximal efficiency. The best IP efficiency was achieved using 3 ␮l of the antimatrix antibodies with 10 ␮l of the concentrated virus stock. Under these conditions, the overall efficiency of the immunoprecipitation was approximately 90%. e B4m1 is a mutant of variant B4 that bears the mutation F(374)Y in the HDV-binding site. f C4m1 is a mutant of variant C4 that bears a V(12)M single mutation in the PreS1 domain of the L envelope protein. g C4m2 is a mutant of variant C4 that bears an N(7)K plus V(12)M double mutation in the PreS1 domain of the L envelope protein.

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FIG 2 Comparison of the HDV binding site sequences of different HBV variants. HDV ribonucleoprotein (HDV RNA genome in complex with approximately 200 molecules of delta antigen [37]) binds to the small cytosolic loop that is located in the S domain, which is present on the L, M, and S envelope proteins of HBV. The amino acid sequence of this HDV binding site (HDVBS) of variant A0 is shown as the reference sequence above the alignment. The amino acid position numbers are given as for the L protein of HBV genotype A. The alignment was performed using MUSCLE alignment software (www.ebi .ac.uk/Tools/msa/muscle/). The variants of HBV (see Table 1) are indicated at the left side of the alignment. The relative values for total yield of HDV virions (total population of released HDV virions, which refers to the sum of all categories of HDV virions regardless of the combination of envelope proteins in the virion) are shown as percentages next to the names of the HBV variants. The absolute numbers of secreted HDV virions were quantified using qPCR as described in Materials and Methods. The highest total yield of HDV virions, facilitated by envelope proteins of variant A0, was used as 100%. The sequences are arranged (from top to bottom) in descending order of efficiency of the HDV assembly. The amino acids that are identical to the reference sequence (HDV-BS of A0) are shown as dashes. The amino acids that are not identical to those of the reference sequence are shown by the single-letter amino acid code. At the bottom of the alignment, the identical amino acid positions are indicated by stars, conservative residues by colons, and a semiconservative residue by a period. The HDV-BS sequence is highly conserved among the variants of HBV. The HDV-BS of B4 bears a unique amino acid change, Y(374)F.

assembly efficiency. However, the mechanism by which the Y(374) residue influences the assembly of HDV is yet to be revealed in the follow-up studies. The total HDV yields (Table 2) reflect the efficiency of interactions of HDV RNP with HBV envelope proteins and the efficiency of egress of the HDV RNP-envelope protein complexes. Given that 24 of 25 variants had highly conserved versions of HDV-BS sequences, it became apparent that the differences in the total HDV yield (with the exception of HDV-B4) could not be attributed to the binding of HDV RNP to

Journal of Virology


HBV envelope proteins and, possibly, could be influenced by specific amino acid residues outside the HDV-BS that may affect the efficiency of the S protein secretion. The virions that contain on their outer side the PreS1 domain of the L protein, which bears the HBV receptor-interacting site, are potentially infectious (16). To access the fraction of the virions that bear the PreS1 domain on the outer surface, we used rabbit polyclonal antimatrix antibodies. Although it was predicted, as described in Materials and Methods, that antimatrix antibodies would be expected to interact exclusively with the PreS1 domain, this was not experimentally examined previously. We therefore tested the specificity of these antibodies. Based on the LMS vector for the D1 variant (LMS-D1), a modified version of this construct was created, in which the initiation codon for the L envelope protein was knocked out (AUG was changed to ACG, which codes for Thr). The resulting construct was therefore designated the L⫺ vector. The parental LMS-D1 vector and the newly made L⫺M⫹S⫹ vector were used to assemble HDV virions. The assembled HDV virions were concentrated with PEG and then were subjected to the IP procedure using either the antimatrix antibodies or the S26 mouse monoclonal antibody. The S26 monoclonal antibody is well studied. It recognizes a very short sequence in the PreS2 domain, QDPR (positions 121 to 124 in the HBV variant D1 sequence) (39). The S26 antibody therefore binds to the L and M envelope proteins. As a negative control, we used HDV virions assembled with the S protein only (HDV-S only virions), which do not bear any PreS sequences. The results are summarized in Fig. 3. As expected, both kinds of antibodies bind HDV-S only virions extremely poorly. The S26 antibody precipitated only 0.5% of the HDV-S only virus particles. Similarly, the antimatrix antibodies pulled down only 0.3% of the HDV-S only virions. Furthermore, the S26 antibody pulled down 30.8% of all HDV-D1 L⫺ virions. This result confirms that a fraction of the assembled virions contained the M protein along with the S (i.e., these virions were L⫺M⫹S⫹). The antimatrix antibodies only precipitated 6.6% of the HDV-D1 L⫺ particles, which is only 21.6% [(6.6/30.8) ⫻ 100] of the virions that were immunoprecipitated by the S26 antibody. This result strongly suggests that the antimatrix antibodies recognize the PreS2 sequences poorly, since they were unable to pull down 78.4% (100 ⫺ 21.6) of L⫺M⫹S⫹ virions that were otherwise available for precipitation (as judged by the virions that were brought down by the S26 antibodies). Next, the HDV-D1 virions that were assembled using the vector that expresses the L, M, and S proteins (LMS-D1 vector) were analyzed. The antimatrix antibodies pulled down 39.6% of all HDV-D1 virions (Fig. 3). The S26 antibody, which recognizes its linear antigenic site on both the L and M proteins, was able to precipitate 34.1% of the same HDV-D1 virions. Considering these data and also taking into consideration the poor ability of the antimatrix antibodies to recognize the M protein (Fig. 3), it became apparent that in the HDV-D1 stock (assembled using the LMS-D1 vector), the majority of the M protein was present in the context of the L⫹ particles and there was no great excess of the L⫺M⫹S⫹ particles. The abovedescribed experiments demonstrated that the maximal percentage of the HDV-D1 L⫺ virions that could be precipitated by the antimatrix antibodies was 6.6%. It can be reasonably assumed that in the context of the HDV-D1 virions assembled with unmodified LMS-D1 vector, which expresses the L, M, and S proteins, the percentage of the L⫺M⫹S⫹ virions that can be precipitated with the antimatrix antibodies (relative to the total number of virions)


FIG 3 Analysis of the specificity of the antimatrix antibodies. Three types of HDV virions were assayed. As a negative control, HDV particles coated with the S envelope protein only were used (S only). The second type of virions was HDV-D1 L⫺ (D1 L-M⫹S⫹). This type of virions was assembled using a construct that was modified from the original LMS-D1 vector that drives the expression of the L, M, and S proteins of variant D1. To make the modified vector, the initiation codon for the L protein was changed from AUG to ACG (ACG codes for Thr). The mutated vector was designated the L⫺ construct (L⫺M⫹S⫹). The virions that were prepared using this construct contained the M and S but not the L protein. The third type of virions was HDV-D1 (D1 L⫹M⫹S⫹); the LMS-D1 vector that expresses the L, M, and S proteins was used for the assembly of this type of virions. All assembled HDV stocks were concentrated with PEG. The concentrated stocks were subjected to immunoprecipitation with the S26 or the antimatrix (aM) antibodies, as indicated below the bars. The S26 mouse monoclonal antibody (a gift from Vadim Bichko) recognizes the short sequence QDPR in the PreS2 domain (39). The S26 antibody binds to the L and M envelope proteins. The antimatrix antibodies (GenScript) are rabbit polyclonal antibodies that were raised against the synthetic peptide that spans positions 91-IPPPASTNRQSGRQPTPISPPLRDS HPQAMQWNSTAFH-128 of the PreS region (HBV genotype A [20]), which incorporates the conserved HBV matrix domain (underlined) (23, 24). Each IP used 10 ␮l of the concentrated virus stock and either 2 ␮l of the S26 antibody or 3 ␮l of the antimatrix antibodies, respectively. The results of the immunoprecipitations are expressed as percentages of the total amount of HDV virions in the stock that was analyzed (y axis). Each bar represents the average of several independent immunoprecipitations. The precipitations of (i) HDV-S only virions with the S26 antibody, (ii) HDV-D1 L⫺ virions with the antimatrix antibodies, and (iii) HDV-D1 virions with the antimatrix antibodies were repeated two times each. The other three immunoprecipitation experiments were repeated three times each. The standard errors of the means are indicated by error bars. The quantification of HDV genomes was done using HDVspecific qPCR (21). During the real-time PCR procedure, each measurement was performed as three independent qPCRs.

cannot exceed the above-described 6.6%. Therefore, the fraction of the PreS1-containing virions among the HDV-D1 virions precipitated with the antimatrix antibodies is expected to be greater or equal to 83.2% [100% ⫺ 16.8%, where 16.8% ⫽ (6.6/39.6) ⫻ 100]. In other words, of the total amount of virions that were immunoprecipitated from the HDV-D1 stock using the antimatrix antibodies, the percentage of the PreS1-HDVs is anticipated to be at least 83.2%. Therefore, the antimatrix antibodies used in the IP procedure were predominantly pulling down the L protein-

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containing (and thus potentially infectious) virions. However, the antimatrix antibodies did not display exclusive specificity for the PreS1 region. They demonstrated preferential specificity for the PreS1 sequences but, at the same time, were able to react with the PreS2 sequences (of the M protein) to a small extent. Overall, we concluded that (i) the predominant majority of the virions immunoprecipitated by the antimatrix antibodies are the PreS1-HDVs and (ii) the contribution of binding to the M envelope protein during IP with the antimatrix antibodies can be considered not significant. Since the antimatrix antibodies displayed predominant but not exclusive specificity for the PreS1-containing virions, the virions pulled down by IP using the antimatrix antibodies were termed PreS1*-HDVs, which can be considered a good acceptable approximation reflecting the fraction of potentially infectious HDV virions. The asterisk is introduced to indicate that the immunoprecipitated virions contain predominantly the PreS1-containing particles, along with nonsignificant amounts of the L⫺M⫹S⫹ virions. The determined absolute amounts of the PreS1*-HDVs varied between different HDV types within an approximately 270-fold range (Table 2). The averaged percentages of PreS1*-HDVs did not exceed 32% of the total number of secreted virions, strongly indicating that the majority of the assembled HDV virions did not contain the L envelope protein (16). The percentages of PreS1*HDVs facilitated by different HBV variants varied within an approximately 3-fold range. The majority of HBV variants supported a yield of PreS1*-HDVs within a range of 21 to 32%, while the highest numbers of 31.9% and 31.8% were observed for HDV-B2 and HDV-D1, respectively. The envelope proteins of HBV genotypes D and E consistently yielded significant fractions of PreS1*-HDVs. The lowest percentages of PreS1*-HDVs were facilitated by the B3 (11.0%), C4 (11.3%), and C5 (11.8%) variants (Table 2). The antimatrix antibodies used for immunoprecipitation were raised against the peptide that spans positions 91 to 128 in the L protein of HBV variant A0. This peptide includes the sequence of the conserved HBV matrix domain (23, 24). Figure 4 represents the alignment of the region spanned by the abovedescribed peptide for all of the sequences used in this study. The majority of amino acids that differ from the A0 sequence represent either conserved or semiconserved changes (Fig. 4). It became apparent that none of the observed amino acid changes seemed to interfere with the IP procedure, since the presence of a particular change or combination of changes did not correlate with the percentage of PreS1*-HDVs determined. Overall, the data generated (Table 2 and Fig. 4) strongly suggest that the antimatrix antibodies recognized similarly the region of amino acid positions 91 to 128 (numbering is for genotype A [20]) in the sequences of the HBV variants that were examined. The following observations can serve as examples of the several lines of supporting evidence for this conclusion. (i) If serious differences existed between different HBV variants in terms of binding to the antimatrix antibodies, then one could expect that the presence of at least some variant-specific amino acid residues that differed from the A0 sequence within the region of positions 91 to 128 (Fig. 4) would correlate consistently with a low percentage of PreS1*-HDVs measured (compared to the results for A0). This would suggest the existence of defect(s) in binding by the antimatrix antibodies. However, according to the data presented in Table 2 and Fig. 4, 19 of 25 variants, which

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FIG 4 Comparison of the sequences at and around the matrix domain of different HBV variants. Rabbit polyclonal antimatrix antibodies raised against the peptide (shown above the alignment) that spans amino acid positions 91 to 128 of the L envelope protein of HBV variant A0 (see Table 1) were used for immunoprecipitation of HDV virions and quantification of the percentages of PreS1*-HDVs (see the text). The sequence of the HBV matrix domain is underlined. The variants of HBV are indicated at the left side of the alignment. The absolute numbers of HDV virions that were immunoprecipitated with the antimatrix antibodies were quantified using qPCR as described in Materials and Methods. The percentages of PreS1*-HDVs in the total population of secreted HDV genome-containing virus particles are indicated next to the variant names. The sequences are presented in descending order (from top to bottom) of the percentage of PreS1*-HDVs, with B2 shown at the top, since it produced the highest percentage of PreS1*-HDVs. The amino acids that are identical to the reference sequence (variant A0) are shown as dashes. The amino acids that are not identical to those of the reference sequence are shown by the single-letter amino acid code. At the bottom of the alignment, the identical amino acid positions are indicated by stars, conservative residues by colons, and a semiconservative residue by a period. The sequence of the matrix domain appeared to be conserved among different HBV variants, as expected.

represent HBV genotypes A to I, displayed percentages of PreS1*HDVs within a limited range of values of between 21 and 32%. (ii) HDV-A0 (the A0 sequence is 100% identical to the peptide that was used to produce the antimatrix antibodies) did not display the highest percentage of PreS1*-HDVs (it displayed 28.0%). Six other viruses were above HDV-A0 in the ranking shown in Fig. 4, displaying very similar but slightly higher percentages of PreS1*-HDVs, as follows: 1, HDV-B2 (31.9% PreS1*-HDVs; has four amino acids that are nonidentical to the above-described peptide sequence of A0); 2, HDV-D1 (31.8%; five nonidentical residues); 3, HDV-I1 (31.0%; two nonidentical residues); 4, HDV-E2 (31.0%; four nonidentical amino acids); 5, HDV-E3 (29.8%; four nonidentical amino acids); and 6, HDV-D5 (29.4%; five nonidentical residues). (iii) Variants B2, B1, and B4 have identical sequences within the fragment spanning positions 91 to 128, bearing four residues that are not identical to the sequence of A0. However, B2 facilitates 31.9% PreS1*-HDVs, while B1 facilitates 22.7% and B4 12.6%. These differences cannot be explained by the sequence differences between the above-named genotype B variants and the A0 variant within the region of positions 91 to 128.

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FIG 5 Detection of delta antigen-positive primary human hepatocytes (PHH) infected in vitro with HDV-A0 (A), HDV-B2 (B), HDV-C3 (C), HDV-D1 (D), HDV-E3 (E), or HDV-F1 (F). At day 9 postinfection, hepatocytes were washed, fixed, permeabilized, and then stained for delta antigen (red), alpha-tubulin (green), and nuclear DNA (blue) as previously described (21, 27). The infection and immunofluorescence procedures are described in Materials and Methods.

(iv) The F1 variant has six nonidentical residues (compared to A0) within the region between positions 91 and 128 and still displayed a very similar percentage of PreS1*-HDVs, 27.3% (for A0, the number was 28.0%). In addition, the IP procedure was optimized, so the antimatrix antibodies were able to pull down the sequences that belonged to different HBV variants with practically equal efficiencies, leading to recoveries of about 90% (and in some cases ⬎90%) of the PreS1*-HDVs (data not shown). The above-described observations indicate that the amino acid residues that define the antigenic sites within the region that is recognized by the antimatrix antibodies likely remain identical or sufficiently conserved among different HBV variants tested. Based on the data summarized in Table 2, no overall HBV genotypespecific trend in regard to either the total yield of HDV or the yield of PreS1*-HDVs was observed. Infectivities of HDV virions coated with the envelope proteins of different HBV variants. The infectivities of the assembled HDVs were assayed using in vitro infection of primary human hepatocytes (PHH). We quantified infected PHH by staining for newly made delta antigens using rabbit polyclonal antibodies raised against recombinant small ␦Ag (21). We also quantified the newly made HDV genomes produced as the result of infection of PHH, by assaying total RNA harvested from infected hepatocytes using qPCR (21, 25). Selected images of PHH infected with different HDV types are presented in Fig. 5, 6, 7, and 8. The origin of the envelope proteins clearly makes a difference in terms of the numbers of HDV-infected cells. For example, HDV-B2 and HDV-D1 virions consistently infected greater fractions of PHH (Fig. 5B and D), while for HDV-A2 and HDV-C2, infected PHH were observed only during one of three and two of four separate infections, respectively. For HDV-C4, no infected cells were observed in three of three infections (Fig. 7B and 8B; also data not shown). There-


fore, HDV-C4 was considered a noninfectious virus. Consistent with HDV replication in PHH, ␦Ag was detected most frequently in the nucleoplasm (Fig. 5 to 8) (21, 27). Some infected hepatocytes displayed a different but also previously observed pattern in which ␦Ag was also found in the cytoplasm (Fig. 6C and D). Since the assembled HDVs did not contain HBV and, therefore, the formation of new virions and HDV spread did not take place during the infection, the appearance of ␦Ag in the cytoplasm is consistent with the occurrence of replication-derived mutations in the ␦Ag nuclear localization signal (21, 37, 40). To describe quantitatively the infectivity of the different HDV types, we employed two parameters, the specific infectivity (SI) and the normalized index of infected PHH (NI). The SI is the number of HDV genomes per hepatocyte produced by infection normalized by the value of the PreS1*-MOI, which is the multiplicity of infection that reflects the number of PreS1*-HDVs in the inoculum used per average hepatocyte. The NI is the percentage of infected PHH normalized by the PreS1*-MOI. The SI and NI values of HDV-C3 were used as 100%. The SI and NI values of other viruses were normalized relative to those of HDV-C3. The results are summarized in Fig. 9. Interestingly, the envelope proteins of different HBV variants supported infectivity of the HDV within a fairly wide range, 160-fold, of SI (%) values. The observed NI (%) values were within an approximately 150-fold range. The most infectious viruses, HDV-C3, HDV-D1, and HDV-B2, demonstrated the highest SI values (81% to 100%) and considerably high NI values (40% to 100%). The next six HDV types, HDV-F1, HDV-D3, HDV-D2, HDV-D5, HDV-B3, and HDV-E3, also displayed relatively high infectivities (SI values within the range of 40% to 49%) while not being as highly infectious as the abovementioned three HDV types. For this group, the NI values ranged between 15% and 39%. The next group of moderately infectious HDVs included HDV-E2, HDV-I1, HDV-C5, HDV-E1, and

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FIG 6 Detection of delta antigen-positive PHH infected in vitro with HDV-G1 (A), HDV-H1 (B), HDV-I1 (C), HDV-D2 (D), HDV-B1 (E), or HDV-C5 (F). Infection of PHH and immunofluorescence assay of infected cells were conducted as described in Materials and Methods. PHH were assayed at 9 days postinfection. Green staining is for alpha-tubulin, red for delta antigen newly synthesized as a result of HDV infection, and blue is DAPI staining for nuclear DNA. (C and D) Note the hepatocytes that displayed staining for delta antigen not only in the nucleoplasm but also in the cytoplasm. These are examples of a less frequent (compared to nucleoplasmic staining) pattern of subcellular localization of delta antigen in infected hepatocytes.

HDV-B1, whose SI and NI values ranged from 27% to 34% and 13% to 49%, respectively. The HDVs coated with envelope proteins of HBV variants G1, B4, A1, A0, and H1 displayed SI values between 9% and 15% and NI values between 10% and 56%. The rest of the HDV types demonstrated low infectivities, with SI val-

ues below 8%. Among them were HDV-A3, HDV-C1, and HDVA4, with SI values in the range of 3% to 8% and NI values between 8% and 12%. HDV-A2, HDV-C2, and HDV-C4 displayed very low SI values (1.6%, 0.6%, and 0.1%, respectively). The observed very low SI values are consistent with the results of the immuno-

FIG 7 Detection of delta antigen-positive PHH infected in vitro with HDV-A1 (A), HDV-A2 (B), HDV-A3 (C), HDV-A4 (D), HDV-B3 (E), or HDV-B4 (F). Infection of PHH and immunofluorescence were conducted as described in Materials and Methods. PHH were analyzed at day 9 after infection. Staining for delta antigens is in red, green is alpha-tubulin, and blue is DAPI staining for nuclear DNA. (B) No infected cells are shown, since no cells positive for delta antigens were observed during two out of three separate infections when PHH were inoculated with HDV-A2.

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FIG 8 Detection of delta antigen-positive PHH infected in vitro with HDV-C1 (A), HDV-C4 (B), HDV-D3 (C), HDV-D5 (D), HDV-E1 (E), or HDV-E2 (F). The details of in vitro infection of PHH and subsequent analysis of HDV-infected cells at day 9 postinfection using immunofluorescence are described in Materials and Methods. Newly synthesized delta antigens are stained in red, green is alpha-tubulin, and blue (DAPI) is nuclear DNA. (B) No cells positive for delta antigens were observed in three of three separate infections when PHH were inoculated with HDV-C4.

fluorescence experiments. As mentioned above, no infected PHH were observed on all occasions for HDV-C4. For HDV-A2 and HDV-C2, infected PHH were observed in one of three and two of four infections, respectively. Therefore, the ability of these virions coated with envelope proteins of variants C2 or A2 to achieve infection of PHH critically depended on the susceptibility of the hepatocytes tested, which varied between different PHH lots. All of the natural M⫺ mutants, A1, B3, C1, and C5, supported the production of infectious HDVs, as expected (41). The envelope proteins of four genotype D variants supported the production of virions with high infectivity (average SI of 59.9%), which was higher than the average SIs for four genotype B variants (41.0%) and three genotype E variants (34.8%), although the average SI for E variants was not dramatically lower than that of the variants of genotype B. All tested variants of genotypes D, B, and E supported the formation of infectious HDVs. The variants of genotype A facilitated the assembly of HDVs with low infectivities (average SI of 6.9%). Therefore, based on the average SI values (Fig. 9) for these HBV genotype-specific envelope proteins, the following trend reflecting support of HDV infectivity was observed: D (59.9%) ⬎ B (41.0%) ⬎ E (34.8%) ⬎ A (6.9%). Within the group of genotype C variants, the SI values were diverse. HDV-C3 had the highest infectivity, while HDV-C5 was moderately infectious. HDV-C1 and HDV-C2 displayed low infectivities, and HDV-C4 was apparently noninfectious. For genotypes F, G, H, and I, only one sequence per genotype was available. HDV-F1, HDV-G1, HDV-H1, and HDV-I1 showed diverse SI levels of between 9.8% and 49%. Another interesting observation was made by comparing the SI and NI values. In Fig. 9, the viruses are placed in descending order of their SI values. It became apparent that the NI values do not always follow the same trend. Thus, there was no direct correla-


tion between the SI and NI values for a number of viruses tested. Several HDV types illustrating the lack of such correlation were next carefully considered, using only the SI and NI values with relatively low standard errors of the means. For example, HDV-B3 seemed to have an NI value that was unreasonably low compared to the corresponding SI value. In other words, HDV-B3 induced a relatively high level of HDV replication in infected PHH while not infecting a considerable number of cells (as normalized by PreS1*MOI). The examples of an opposite tendency are HDV-F1, HDVD3, HDV-I1, HDV-C5, HDV-B1, HDV-G1, HDV-B4, and HDVH1. They demonstrated NI values that were higher than could be anticipated based on the corresponding SI values. These virions seemed to infect sufficient numbers of PHH, which did not, however, result in respectively high levels of HDV replication. Apparently, the infection of considerable numbers of hepatocytes does not guarantee high levels of HDV replication. The implications of these findings are further evaluated in Discussion. These interpretations are in agreement with the results of testing the infectivity of the B4 mutant bearing the single change F(374)Y. Compared to the SI and NI values of HDV-B4, HDV coated with mutated B4 envelope proteins (HDV-B4m1) displayed an approximately 3-fold higher SI value (34.3% versus 12.2%) while exhibiting a similar NI value (65.0% versus 55.9%) (Fig. 9). The data suggest that there was definitely an increase in the number of HDV genomes that started HDV RNA replication after successful entry, while there was no significant increase in the number of infected cells. One possible explanation is that the mutation introduced was important for the coordinated mechanism of HDV RNP disassembly from the envelope proteins during the postentry intracellular trafficking and had no effect on entry per se. Finally, we investigated why HDV-C4 appeared noninfectious. Only two unique substitutions, M(12)V and K(7)N, were identi-

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fied for the C4 variant in the critical for infectivity PreS1 domain (Fig. 10A). While the significance of the former substitution was not obvious, the latter, according to analysis using the software web.expasy.org/myristoylator/, could affect the functionality of the myristoylation signal. Two new types of HDV virions were produced. The envelope of HDV-C4m1 contained the mutated version of the L protein of the C4 variant, bearing the V(12)M change. In HDV-C4m2, the L envelope protein of the C4 variant contained two amino acid changes, V(12)M and N(7)K. The assembled concentrated virus stocks of HDV-C4m1 and HDVC4m2 did not differ significantly from HDV-C4 in terms of the total yield of virions or the percentage of the PreS1*-HDVs (Table 2). Both newly made HDV types were demonstrated to be infectious. The images of the infected PHH are shown in Fig. 10B. A V(12)M single mutation led to the observation of infected PHH (NI of 5.8%), while no improvement in the SI was detected. The double mutant appeared more infectious than HDV-C4m1 and HDV-C2 (Fig. 9). However, the NI and SI values of HDV-C4m2 were still considerably lower than those of the more infectious HDV-C3 and HDV-C5. The results suggest that, while positions 7 and 12 were critical for infectivity, the overall infectivity likely was additionally regulated by other amino acid residue(s) outside the PreS1 region. DISCUSSION

FIG 9 Infectivity parameters of different types of HDV virions, coated with envelope proteins of different HBV variants belonging to nine genotypes of HBV (A to I). Two parameters that define the infectivity of the different types of HDV virions, the specific infectivity (SI) and normalized index of infected cells (NI), were determined at day 9 postinfection. SI is the number of HDV genomes per cell, which were produced and accumulated during infection of PHH, normalized per each PreS1*-HDV virion (see the text) used in the inoculum per average hepatocyte. The NI represents the percentage of HDV-positive PHH normalized by the number of PreS1*-HDVs per cell in the inoculum used. For SI quantification measurements, multiplicities of infection (MOI) of 10 to 20 total genome equivalents (GE) of HDV/hepatocyte were used. For immunofluorescence experiments, MOI of 30 to 100 HDV GE/cell were used. The data shown are based on the results of independent infections (each using a different lot of PHH) and summarize the outcomes of (i) two independent infections for HDV-A3, HDV-B1, HDV-B2, HDV-B3, HDV-D2, HDV-D3, and HDV-G1; (ii) three independent infections for HDV-A1, HDV-A2, HDVA4, HDV-C1, HDV-C3, HDV-C4, HDV-D5, HDV-E1, HDV-E2, HDV-E3, HDV-H1, and HDV-I1; (iii) four for HDV-A0, HDV-B4, and HDV-C2; (iv) five for HDV-C5, and HDV-F1; and (v) eight for HDV-D1. The mutants of variant C4 [C4m1, bearing the single change V(12)M, and C4m2, harboring the two changes N(7)K and V(12)M] were compared to their wild-type counterpart in one independent infection. The mutant of B4 [B4m1, bearing the mutation F(374)Y] was compared to wild-type B4 in another independent infection. Within each infection (a single individual lot of PHH), the SI value for each HDV type was determined based on three independent repeats (three different wells of infected PHH), while the immunofluorescence assay was conducted using two different wells of infected PHH (i.e., two independent repeats). The SI and NI values shown are the averages. The standard errors of the means are indicated by error bars. The HDV types used as the inocula are named according to the HBV variant whose envelope proteins were used for HDV assembly. For example, C3 stands for HDV virions assembled with the envelope proteins of HBV genotype C, variant number 3. The data are arranged from top to bottom in descending order of the SI values. An example of a variant (HDV-B3) whose calculated NI value appeared to be lower than anticipated based on the corresponding SI value is marked with an asterisk. Several examples of variants whose NI values were higher than anticipated based on their corresponding SI values are marked with two asterisks.

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Our study contributes to the understanding of the complex mechanisms that define the infectivity of HBV and HDV virions. Since both viruses have indistinguishable envelopes (16), they share the same receptor and steps of attachment and entry that are regulated by HBV envelope proteins. The HDV model allows us to study the contribution of HBV envelope proteins to overall infectivity separately, in the absence of other HBV components. This is the first report that demonstrates that HDV of genotype I (26, 42) is fully compatible with the nine HBV genotypes A to I in terms of assembly and infectivity. This finding suggests that, although HDV genotypes are not uniformly distributed around the world (43, 44), at least for HDV genotype I, there likely will be no restrictions in terms of support of (i) the transmission and (ii) the development of infection by different HBV genotypes. Our data, therefore, not only provide new insights into the understanding of HDV-HBV interactions but have a certain clinical value regarding the transmission of HDV disease and its relation to the geographical distribution of HBV genotypes. Out of 25 HBV variants that were examined, isolates A2, B1, B2, B4, C2, C5, D1, D2, E1, E3, G1, and H1 corresponded to the chronic stage of HBV infection, while A1, B3, C1, and I1 were collected during acute HBV infection (17, 28–36; K. Abe, personal communication). Corresponding information regarding the rest of the variants is not available. The small number of isolates from the acute stage of infection that were analyzed is insufficient to form conclusions regarding the extent of infectivity supported by the envelope proteins expressed during acute infection versus that of the chronic phase of infection. Based on our data that the envelope proteins from the variants collected during chronic infection facilitated the production of HDV types with a wide variety of infectivity parameters (both NI and SI) (Fig. 9), including several of the most infectious HDV types, it cannot be asserted that at the chronic stage of infection, HBV envelope proteins are responsible for assembly of mainly virus particles with low infectivity that do not support efficient virus transmission. Further studies, which

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FIG 10 Analysis of two mutants of variant C4. In vitro infections of PHH with HDV-C4 resulted in an absence of delta antigen-positive cells on three separate occasions (Fig. 8B). The PreS1 domain, especially the region between positions 12 and 86 (this numbering is for genotypes that have an additional 11-amino-acid extension at the N terminus of PreS1, which results in a 119-amino-acid-long PreS1 domain), was demonstrated previously as being critical for HBV and HDV infectivity (23). (A) Alignment of the PreS1 domains of variants C1 to C5. The top row displays the sequence of the PreS1 domain of C3 (positions 1 to 119) in single-letter code. For the other HBV genotype C variants, nonidentical amino acids are shown as the corresponding single letters and identical amino acids are shown as dashes. At the bottom of the aligned sequences, stars correspond to identical residues, colons mark conserved residues, and periods mark semiconserved amino acids. In its PreS1, C4 has only two unique changes, K(7)N and M(12)V (underlined), that were not found in any other sequences tested in this study (Table 1). A third change, I(84)L, which was found only in C4 and not in other genotype C sequences, was also present in variant B4, which facilitated the production of infectious HDV (Fig. 9). Two mutants of C4 were made, C4m1, bearing the single reverse change V(12)M, and C4m2, harboring the two reverse changes N(7)K and V(12)M. (B) An immunofluorescence analysis of PHH infected with either HDV-C4m1 or HDV-C4m2 was performed to assay their levels of infectivity. The infection procedure and immunofluorescence assay (at 9 days postinfection) were conducted as described in Materials and Methods. The red staining represents delta antigens, blue is DAPI staining of nuclear DNA, and alpha-tubulin is stained in green. The image on the right side displays staining for delta antigens not only in nuclei but also in the cytoplasm. The quantitative results of the infectivity tests (SI and NI values) are summarized in Fig. 9.

will employ larger numbers of samples from acute and chronic stages of HBV infection, as well as different HBV strains collected from the same host, are warranted in order to better understand the mechanisms of (i) maintenance of chronic HBV infection, (ii) HDV-HBV interactions, and (iii) dependence of HDV persistence on the variety of different HBV sequences that coexist in a particular host. It was observed that the envelope proteins of several HBV variants ensured relatively high numbers of infected PHH and high levels of overall replication (variants C3, D1, and B2). Other variants (for example, F1, D3, I1, C5, B1, G1, B4, and H1) supported levels of HDV replication (SI values) that were nonproportionally lower than could be anticipated based on the corresponding NI values (normalized numbers of infected PHH). In other words, infection of sufficient numbers of PHH did not always ensure overall high levels of HDV replication. Another variant, B3, facilitated a considerable level of replication with an unexpectedly low NI value. Therefore, since all types of HDV virions tested had the same HDV RNP inside and differed only by the variant-specific envelope proteins coating the particle, it became apparent that the HBV envelope proteins alone determined two parameters: (i) the


number of infected cells and (ii) the number of HDV genomes that successfully started replication after entry. One interpretation is that HBV envelope proteins not only facilitate attachment and entry but also regulate at least one postentry step of the virus life cycle (i.e., likely one or more events related to intracellular trafficking). Our data further suggest that HBV and HDV likely employ endocytosis as the entry mechanism, as was also suggested by other laboratories (45, 46). The entry via endocytosis explains how the envelope proteins determine the number of viral genomes that successfully reach the replication site(s) and start replication. It needs to be further explored whether the determinants of the envelope proteins that regulate the number of infected cells and the number of genomes that will succeed in initiation of the replication are structurally and functionally separated. The kinetics of virus spread throughout the liver, as was previously noted by Asabe et al., likely determines the timing and magnitude of the host immune response, which in turn will determine whether infection will become transient or chronic (3). The ability of HBV envelope proteins to determine the number of infected cells and the fraction of genomes that will initiate replication will likely have a considerable impact on defining the kinetics of virus

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spread throughout the liver. This is likely true for both HDV and HBV, since both viruses share the same envelope proteins. Our data suggest that the kinetics of the spread of virus through the liver can be tightly linked to the overall infectivity of the virions. Therefore, it is reasonable to consider the infectivity as a critical determinant of the virus-induced pathogenesis, which is likely involved in determining the outcome (transient or chronic) of either HBV or HDV infection. Considering the impact of infectivity on the kinetics of virus spread, it is interesting to hypothesize that even an HDV variant that has a relatively low rate of replication may achieve a state of persistent infection when the spread of HDV is supported by the envelope ensuring a high number of infected hepatocytes. The observed trend for HBV genotypes in terms of supporting HDV infectivity, D (59.9%) ⬎ B (41.0%) ⬎ E (34.8%) ⬎ A (6.9%) (based on the average SI values) (Fig. 9), may advocate in favor of follow-up studies using larger numbers of tested HBV variants. The data generated also suggest that genotyping of HBV could be helpful in anticipating the outcomes of HBV and HBV/HDV infections.

12.

13.

14. 15. 16. 17. 18.

ACKNOWLEDGMENTS S.O.G. and S.M. were supported by NIH grant NCI R01CA166213. S.O.G. was also supported by NIH grants NIAID R21AI097647 and NCRR P20RR016443 and by the University of Kansas Endowment Association. We are grateful to Stefan Wieland, Frank Chisari, Hans Will, Volker Bruss, Yu-Mei Wen, and Stephan Schaefer, who provided us with constructs bearing sequences of different HBV variants. We thank Vadim Bichko for his generous gift of the S26 monoclonal antibody. We acknowledge the help of Jessica Salisse, Megan Dudek, and Louise Rodrigues. We thank Igor Prudovsky for constructive comments.

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